One of the goals of reconstructive surgery is to be able to replace damaged tissue with new tissue, perhaps grown from a patient""s own cells. For example, researchers have endeavored to develop cartilage regeneration systems in which isolated chondrocytes are injected into a damaged area in the context of a polymer scaffold (see, for example, Atala et al., J. Urol. 150:747, 1993; Freed et al., J. Cell. Biochem. 51:257, 1993 and references cited therein). Similar seeded scaffold systems have been studied in the context of bone repair, where osteoblast cells are utilized in conjunction with polymeric or ceramic supports (see, for example, Elgendy et al., Biomater. 14:263, 1993; Ishaug et al., J. Biomed. Mater. Res. 28:1445, 1994). Seeded compositions have also been studied for their utility in bladder control and vesicoureteral applications (see, for example, Griffith-Cima et al., published PCT application no. WO 94/25080.
Researchers in the field have identified several characteristics that are desirable for scaffold materials to be used in such seeded compositions. For example, Freed et al. (Bio/Technology 12:689, 1994) list the following six factors as desirable features:
(1) the scaffold surface should permit cell adhesion and growth;
(2) neither the scaffold material nor its degradation products should provoke inflammation or toxicity when implanted in vivo;
(3) the scaffold material should be reproducibly processable into three dimensional structures;
(4) the scaffold material should have a porosity of at least 90% so that it provides high surface area for cell-scaffold interactions, sufficient space for extracellular matrix regeneration, and minimal diffusion constraints during in vitro culture;
(5) the scaffold material should resorb once it has served its purpose of providing a template for the regenerating tissue; and
(6) the scaffold degradation rate should be adjustable to match the rate of tissue regeneration by the cell type of interest.
Much effort has been spent in attempts to identify materials that can act as effective scaffolds for tissue repair. There remains a need for the development of suitable new materials for use as scaffolds in cell seeding applications.
xe2x80x9cAmorphousxe2x80x9dxe2x80x94By xe2x80x9camorphousxe2x80x9d as that term is used here, it is meant a material with significant amorphous character. Significant amorphous character contemplates greater than 75% amorphous content, preferably greater than 90% amorphous content, and is characterized by a broad, featureless X-ray diffraction pattern. It is recognized that a small degree of crystallinity may exist in the material. However, for the amorphous precursor materials of the present invention, it is preferable that the degree of crystallinity be less than that desired in the product material.
xe2x80x9cBioactivexe2x80x9dxe2x80x94xe2x80x9cBioactivexe2x80x9d refers to a material that induces hard tissue formation in and about the implant. When implanted in soft tissue, the bioactivity may also require the presence of a growth or trophic factor, or the seeding of the implant with a hard tissue forming cell type.
xe2x80x9cBiocompatiblexe2x80x9dxe2x80x94The term xe2x80x9cbiocompatiblexe2x80x9d, as used herein, means that the material does not elicit a substantial detrimental response in the host. There is always concern, when a foreign object is introduced into a living body, that the object will induce an immune reaction, such as an inflammatory response that will have negative effects on the host. For example, although hydroxyapatite is generally considered to be xe2x80x9cbiocompatiblexe2x80x9d, significant inflammation and tissue necrosis have been observed when crystalline hydroxyapatite microcarriers are inserted intramuscularly in animals (see, for example, IJntema et al., Int. J. Pharm 112:215, 1994).
xe2x80x9cBioresorbablexe2x80x9dxe2x80x94xe2x80x9cBioresorbablexe2x80x9d refers to the ability of a material to be resorbed in vivo. xe2x80x9cFullxe2x80x9d resorption means that no significant extracellular fragments remain. The resorption process involves elimination of the original implant materials through the action of body fluids, enzymes or cells. Resorbed calcium phosphate may, for example, be redeposited as bone mineral, or by being otherwise reutilized within the body, or excreted. xe2x80x9cStrongly bioresorbablexe2x80x9d, as that term is used herein, means that at least 80% of the total mass of material implanted intramuscularly or subcutaneously is resorbed within one year. In preferred embodiments of the invention, the strongly resorbing PCA calcium phosphate is characterized in that, when at least 1 g (preferably 1-5 g) of PCA material is implanted at a subcutaneous or intramuscular site, at least 80% of the material is resorbed w/in one year. In more preferred embodiments, the material will be resorbed within nine months, six months, three months, and ideally one month. Furthermore, particularly preferred materials are characterized in that they can be fully resorbed in the stated time periods. For the purpose of this disclosure, xe2x80x9cweaklyxe2x80x9d resorbable means that less than 80% of the starting material is resorbed after one year.
xe2x80x9cCellsxe2x80x9dxe2x80x94the term xe2x80x9ccellsxe2x80x9d, as used herein, refers to any preparation of living tissue, including primary tissue explants and preparations thereof, isolated cells, cells lines (including transformed cells), and host cells.
xe2x80x9cEffective Amountxe2x80x9dxe2x80x94An effective amount of a biologically active agent is an amount sufficient to elicit a desired biological response.
xe2x80x9cHardeningxe2x80x9dxe2x80x94xe2x80x9cHardeningxe2x80x9d refers to the process by which the hydrated precursor is transformed into a hardened PCA material. The PCA material is considered to be xe2x80x9chardenedxe2x80x9d when it is a substantially non-formable solid. Such a hardened PCA material has minimal compressibility and tends to undergo plastic as opposed to elastic deformation.
xe2x80x9cHydrated precursorxe2x80x9dxe2x80x94The term xe2x80x9chydrated precursorxe2x80x9d, as used herein, refers to the paste or putty formed by hydration of the dry PCA precursors in the presence of a limited amount of aqueous solution (i.e., less than approximately 1 mL aqueous solution/1 g precursor powder). The hydrated precursor may comprise both reactants and products, in various combinations, depending on the extent to which the conversion has progressed. Both the xe2x80x9cinjectablexe2x80x9d and xe2x80x9cformablexe2x80x9d PCA precursor pastes described herein are hydrated precursors. Preferred xe2x80x9cinjectablexe2x80x9d hydrated precursors have a consistency appropriate for delivery through an 18 gauge needle.
xe2x80x9cPoorly crystalline apatitic calcium phosphatexe2x80x9d, xe2x80x9cPCA calcium phosphatexe2x80x9d and xe2x80x9cPCA materialxe2x80x9d, as those terms are used herein, describe a synthetic poorly crystalline apatitic calcium phosphate. The PCA material is not necessarily restricted to a single calcium phosphate phase provided it has the characteristic XRD and FTIR pattern. A PCA calcium phosphate has substantially the same X-ray diffraction spectrum as bone. The spectrum is generally characterized by only two broad peaks in the region of 20-35xc2x0 with one centered at 26xc2x0 and the other centered at 32xc2x0. It is further characterized by FTIR peaks at 563 cmxe2x88x921, 1034 cmxe2x88x921, 1638 cmxe2x88x921 and 3432 cmxe2x88x921 (xc2x12 cmxe2x88x921). Sharp shoulders are observed at 603 cmxe2x88x921 and 875 cmxe2x88x921, with a doublet having maxima at 1422 cmxe2x88x921 and 1457 cmxe2x88x921.
xe2x80x9cPromoterxe2x80x9dxe2x80x94The term xe2x80x9cpromoterxe2x80x9d, as used herein, describes a material or treatment that promotes hardening of a hydrated precursor and may enhance the ACP to PCA calcium phosphate conversion. Some promoters participate in the conversion and are incorporated into the product PCA material; others, known as xe2x80x9cpassivexe2x80x9d promoters, do not participate.
xe2x80x9cReactivexe2x80x9dxe2x80x94xe2x80x9cReactivexe2x80x9d is used herein to refer to the ability of an amorphous calcium phosphate when mixed with liquid to form a hydrated precursor to undergo conversion to the PCA material of the present invention in the presence of a promoter in association with hardening of the precursor materials. Preferred ACPs are characterized by an ability to convert completely, an ability to convert quickly with hardening, an ability to undergo conversion with otherwise inert compounds and/or an ability to convert into a substantially homogeneous PCA material. Where the ACP is reacted with a second calcium phosphate, the xe2x80x9cconversionxe2x80x9d can encompass conversion of both the ACP and the second calcium phosphate. The degree of hardening and the kinetics of the hardening process are also important elements of reactivity. Some ACPs are more reactive than others. An ACP is considered xe2x80x9chighly reactivexe2x80x9d if it undergoes hardening in conjunction with conversion to a PCA material in the presence of a weak promoter, such as dicalcium phosphate dihydrate (xe2x80x9cDCPDxe2x80x9d) with a grain size distribution containing a significant fraction of grain greater than 100 xcexcm. Preferred highly reactive ACPs produce a hardened PCA material in the presence of weakly promoting DCPD and water at 37xc2x0 C. in less than twelve hours, with hardening being substantially complete in about one to five hours, and ideally 10-30 minutes.
The present invention provides a synthetic, poorly crystalline apatitic (PCA) calcium phosphate material that has excellent biocompatibility, resorbability, and processability characteristics. The PCA calcium phosphate of the present invention is useful as a scaffold in any of a variety of in vivo and in vitro cell seeding applications.
The synthetic PCA material of the present invention is prepared in a two-step process in which i) at least one amorphous calcium phosphate (ACP) is exposed to a promoter in the presence of a limited amount of aqueous solution (preferably buffered to ensure compatibility with living cells), so that a hydrated precursor is formed; and ii) the hydrated precursor is allowed to harden, with concomitant conversion of the ACP (and any other reactants) to the synthetic PCA material. The reaction conditions employed to form the PCA material of the present invention are mild, so that the material (in either hydrated or hardened form) is compatible with living cells. Cells may be introduced into the material either at the hydrated precursor stage or after the material has hardened.
The PCA material of the present invention is strongly resorbable in vivo. At least 80% of the total mass of PCA material implanted intramuscularly or subcutaneously is resorbed within one year. More preferably, the PCA material is formulated so that at least 80% of an implant comprising at least 1 g of material, and preferably at least 1-5 g of material, is resorbed within one year. Still more preferably, the PCA material is formulated so that such an implant is resorbed within 9 months, 6 months, 3 months, or, ideally, 1 month. It will be appreciated, however, that resorption is related to surface area, so that the observed resorption of the inventive PCA material may vary according to the conformation of the material (e.g., as a disc, rod, plate, or other three-dimensional structure).
The PCA material of the present invention is highly formable and processable, particularly at the hydrated precursor stage, which is typically formulated as a paste or putty. The material may be formed into any of a variety of useful shapes, before or after cell seeding, and can be delivered to the site of use by any of a variety of methods. For in vivo applications, the material may be hardened in vitro (preferably at an elevated temperaturexe2x80x94at or above about 37xc2x0 C.) and subsequently introduced into an animal or human subject, e.g., by surgical implantation. Alternatively, the PCA material may be introduced into the body in hydrated precursor form and allowed to harden in situ. In preferred embodiments, the PCA material of the present invention substantially hardens in vivo within 15-40 minutes.
The present invention therefore provides therapeutic, structural, or cosmetic implants comprising the inventive PCA material and at least one cell. Preferably, the at least one cell is a bone-forming or bone-degrading cell. Particularly useful cell types include chondrocytes, osteocytes, osteoblasts, osteoclasts, mesenchymal stem cells, fibroblasts, muscle cells, hepatocytes, parenchymal cells, cells of intestinal origin, nerve cells, and skin cells, and may be provided as primary tissue explants, preparations of primary tissue explants, isolated cells, cell lines, transformed cell lines, and host cells. The implants may also comprise additional components such as biologically active agents or factors that alter the characteristics (such as resorbability, strength, adherence, injectability, frictional characteristics, etc.).
The invention also provides methods of preparing such implants; methods of growing bone or cartilage in vivo or in vitro, at natural sites or ectopic sites; methods of osseous augmentation; and methods of diagnosing disease states by assaying tissue-forming potential of cells isolated from a host. The invention also provides in vitro cell culture systems and cell encapsulation matrices.